Program Director/Principal Investigator (Last, first, middle): Wang, Kevin, Yueju A stroke occurs when a blood clot or a narrowing of blood vessels prevents blood from getting to the brain. Globally, stroke is the second leading cause of death among people over the age of 60 years. It is also a major source of serious, long-term disability. The only clot- busting drug approved for the treatment of acute ischemic stroke (AIS) disease is a recombinant human tissue plasminogen activator (rt-PA) obtained from Chinese Hamster Ovary cell (CHO) lines. However, yields of rt-PA produced in CHO cells are relatively low, while the dose of rt-PA required for treatment is high, making this therapy very expensive for developing countries. Therefore, a more efficient method of providing rt-PA would greatly reduce the cost and beof great benefit to patients suffering from acute ischemic stroke disease. The objective of the proposed project is to develop a low-cost, plant seed-based platform for the large scale production of functional t-PA for treatment of acute ischemic stroke patients. Our preliminary data revealed that overexpression of several blood-clot dissolving serine protease genes in plant leaves resulted in death of the leaf tissue. In contrast, our preliminary data also indicated that a recombinant vampire bat plasminogen activator (DSPA?1) could be reliably produced in seeds with sufficient activity to dissolve human blood clots. In this project, appropriate plant expression vectors will be developed to target t-PA gene expression and production to seeds in order to obtain high levels of expression and increased stability of the end product. Transgenic rt-PA plant lines will be generated and in vitro evaluation of seed- derived rt-PA, such as fibrin degradatin and human clot-dissolving assay, will be performed. The overall goal is to develop a seed-based platform in plants for potential large scale, low- cost production of functional rt-PA for the treatment of AIS in humans. 1 Research Strategy